Lentinan-gold nanocluster fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a lentinan-gold nanocluster fluorescent probe as well as a preparation method and application thereof, and relates to the technical field of biomedical materials and natural polysaccharide functionalization. Aminated lentinan and glutathione modified gold nanoclusters are used as raw materials, and covalent coupling is performed under mild conditions to obtain the lentinan-gold nanocluster fluorescent probe. Strong acid, strong alkali and high temperature conditions are avoided in the preparation process, and the natural triple helix conformation of the lentinan can be effectively reserved, so that the inherent anti-tumor biological activity of the lentinan is maintained. The prepared fluorescent probe is uniform in particle size, the average size is about 150 nm, and tumor site enrichment can be achieved through the enhanced permeation and retention effect (EPR effect) of tumors. In a tumor microenvironment, the inherent anti-tumor activity of the lentinan and the tumor cell toxicity induced by peroxidase-like activity shown by Au-GSH generate a synergistic effect, and the probe has a stable fluorescence signal and can realize in-vivo tracing and imaging.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and functionalization of natural polysaccharides, specifically to a lentinan-gold nanocluster fluorescent probe, its preparation method, and its application. Background Technology

[0002] Lentinan (LNT) is a natural β-glucan derived from shiitake mushrooms. It possesses well-defined immunomodulatory, antitumor, and anti-inflammatory biological activities and has been extensively studied and partially applied in clinical adjuvant therapy. However, lentinan itself lacks imaging capabilities, making it difficult to visualize and monitor its distribution, metabolism, and targeted accumulation processes in vivo, thus limiting its further application in precision medicine and integrated tumor diagnosis and treatment.

[0003] Studies have shown that the bioactivity of lentinan (shiitake mushroom polysaccharide) is highly dependent on its natural triple helix structure, which is the important structural basis for its immune-enhancing and anti-tumor effects. However, existing polysaccharide modification or nanostructuring methods often require harsh reaction conditions, such as strong acids, strong alkalis, or high temperatures, which easily destroy the triple helix conformation of polysaccharides, leading to a significant decrease in bioactivity. Therefore, how to effectively preserve the triple helix structure of lentinan while achieving functionalization modification is a key technical problem that urgently needs to be solved in this field.

[0004] Gold nanoclusters have shown promising applications in bioimaging and tumor therapy due to their excellent fluorescence properties, biocompatibility, and peroxidase-like activity. In particular, glutathione-modified gold nanoclusters (Au-GSH) not only possess stable fluorescence signals but can also catalyze the generation of reactive oxygen species in the tumor microenvironment, thereby inducing tumor cell damage. However, Au-GSH particles are only about 2 nm in size, lacking tumor accumulation capacity and exhibiting limited in vivo accumulation efficiency.

[0005] Therefore, developing a novel preparation method that can effectively combine Au-GSH with lentinan under mild conditions, preserving the triple helix structure and bioactivity of lentinan while endowing it with fluorescent tracer and synergistic anti-tumor functions, is of great significance for promoting the application of natural polysaccharides in the integrated diagnosis and treatment of tumors. Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a lentinan-gold nanocluster fluorescent probe, its preparation method and application, specifically involving a method for preparing a lentinan-gold nanocluster fluorescent probe with lentinan as the main body and gold nanoclusters as the fluorescence and catalytic units, which is suitable for biomedical applications such as tumor imaging, tumor treatment and integrated diagnosis and treatment.

[0007] This invention provides a method for preparing a lentinan-gold nanocluster fluorescent probe. The method involves combining gold nanoclusters (Au-GSH) with LNT-NH2 to obtain an LNT-Au fluorescent probe. This method effectively preserves the triple-helix structure and bioactivity of lentinan, while simultaneously endowing the probe with fluorescent tracking function and tumor enrichment ability, showing broad application prospects in tumor diagnosis and treatment.

[0008] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing a lentinan-gold nanocluster fluorescent probe, comprising the following steps: A lentinan-gold nanocluster fluorescent probe was prepared by covalently coupling the amino-modified lentinan and glutathione-modified gold nanoclusters under mild conditions.

[0009] In one specific embodiment of the present invention, the mass ratio of the amino-modified lentinan to the glutathione-modified gold nanoclusters is 0.8~1.2:0.8~1.2.

[0010] In one specific embodiment of the present invention, the preparation method of the aminoated lentinan includes the following steps: Lentinan was dissolved in an organic solvent to obtain a lentinan solution, and the pH of the lentinan solution was adjusted to 8.5-9. Succinic anhydride was dissolved in an organic solvent to obtain a succinic anhydride solution; the succinic anhydride solution was added to the lentinan solution for reaction, and the pH of the system was maintained at 8.5-9 during the reaction. After the reaction was completed, the pH of the system was adjusted to 6.5-7. The product was dialyzed, concentrated, precipitated, washed and dried to obtain LNT-COOH. The LNT-COOH was dissolved in an organic solvent, and then HATU, HOBT and DIEA were added to react. H2N-NHBOC was then added to carry out an amination reaction. After the reaction was completed, trifluoroacetic acid was added and stirred. The product was dialyzed, concentrated and dried to obtain amination-modified lentinan.

[0011] In one specific embodiment of the present invention, in the preparation method of the aminated lentinan, the mass ratio of lentinan to succinic anhydride is 3-5:1, the organic solvent is DMSO, and the molar ratio of HATU, HOBT, DIEA and H2N-NHBOC is 0.1:0.1:0.2:0.6.

[0012] In one specific embodiment of the present invention, the method for preparing the glutathione-modified gold nanoclusters includes the following steps: HAuCl4 H2O was dissolved in water, and glutathione was added to produce a microemulsion suspension. After vigorous stirring, NaBH4 solution was quickly added and stirring continued. The product was then filtered, purified, and concentrated to obtain glutathione-modified gold nanoclusters.

[0013] In one specific embodiment of the present invention, in the method for preparing the glutathione-modified gold nanoclusters, HAuCl4 The molar ratio of H2O, glutathione and NaBH4 is 1.2~1.4:4.8~5.6:12~14, more preferably 1.3:5.2:13.

[0014] In one specific embodiment of the present invention, the preparation method includes the following specific steps: Glutathione-modified gold nanoclusters were dissolved in dimethyl sulfoxide to obtain an Au-GSH solution. A condensing agent, an auxiliary agent, and an alkaline catalyst were added sequentially to the Au-GSH solution. After stirring and activation, amino-modified lentinan was added to carry out the reaction. After the reaction was completed, the solution was dialyzed, concentrated, and freeze-dried to prepare a lentinan-gold nanocluster fluorescent probe.

[0015] In one specific embodiment of the present invention, the concentration of the Au-GSH solution is 1.5~2.5 mg / mL.

[0016] In one specific embodiment of the present invention, the molar ratio of the condensing agent, the auxiliary agent and the alkaline catalyst is 0.1:0.1:0.2.

[0017] In one specific embodiment of the present invention, the condensing agent is HATU, the auxiliary agent is HOBT, and the alkaline catalyst is DIEA; The dialysis was performed using a dialysis bag with a molecular weight cutoff of 50,000 Da.

[0018] In one specific embodiment of the present invention, the reaction time for adding the aminoated lentinan is 1.5 to 2.5 hours.

[0019] One specific embodiment of the present invention discloses a method for preparing a lentinan fluorescent probe, comprising the following steps: Step 1: Dissolving Au-GSH: Dissolve 100 mg of concentrated Au-GSH in 50 mL of DMSO and stir until completely dissolved to ensure that Au-GSH is fully dispersed in the solvent.

[0020] Step 2: Add HATU, HOBT, and DIEA: Add 38.31 mg (0.1 mmol) HATU, 14.3 mg (0.1 mmol) HOBT and 26.04 mg (0.2 mmol) DIEA to the solution in sequence, stir for 3 min to ensure that each reagent is completely dissolved and reacts fully.

[0021] Step 3: Add LNT-NH2: Add 100 mg LNT-NH2 to the reaction solution and stir for 2 h to ensure that the aminoated lentinan is fully combined with Au-GSH.

[0022] Step 4: Dialysis and Concentration After the reaction was complete, the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 50,000 Da to remove unreacted reagents and small molecule impurities. The dialysis time was 72 h. After dialysis, the solution was concentrated.

[0023] Step 5: Freeze-drying The concentrated solution was freeze-dried to obtain the lentinan fluorescent probe (LNT-Au).

[0024] Furthermore, in step one, during the Au-GSH dissolution process, ensure that the volume of DMSO used is 50 mL to guarantee complete dissolution and maintain a homogeneous reaction solution. The reaction temperature should be controlled at room temperature, and the stirring time should be 30 minutes.

[0025] Furthermore, in step two, when adding HATU, HOBT, and DIEA, ensure that the amount of each reagent added is accurate, and that the molar ratio of HATU to HOBT to DIEA is 0.1:0.1:0.2.

[0026] Furthermore, in step three, when adding the aminated lentinan LNT-NH2, ensure that its mass ratio to Au-GSH is 1:1, and the reaction time should be 2 hours. During the reaction, maintain the temperature at room temperature and ensure thorough stirring to promote complete reaction.

[0027] Furthermore, in step four, the dialysis time should be 72 hours, using a dialysis bag with a molecular weight cutoff of 50,000 Da to ensure thorough removal of unreacted reagents and low molecular weight impurities. After dialysis, the solution should be concentrated at a concentration ratio of 1:5.

[0028] Furthermore, in step five, the freeze-drying temperature should be set to -80℃, and the drying time should be 48 hours to ensure the purity and stability of the final product. During the freeze-drying process, care should be taken to avoid excessively high temperatures to prevent affecting the properties of the probe.

[0029] Secondly, the present invention provides a lentinan-gold nanocluster fluorescent probe, which is obtained by the preparation method described above.

[0030] The LNT-Au fluorescent probe prepared in this invention retains the natural triple helix conformation of lentinan, thereby maintaining its inherent antitumor biological activity. It has a uniform particle size with an average size of about 150 nm, which is conducive to the enrichment of tumor sites through the enhanced penetration and retention effect (EPR effect) of tumors. In the tumor microenvironment, the inherent antitumor activity of lentinan can synergistically work with the tumor cell cytotoxicity induced by the peroxidase-like activity exhibited by Au-GSH. At the same time, the probe has a stable fluorescence signal, which can realize in vivo tracking and imaging.

[0031] In one specific embodiment of the present invention, the average particle size of the lentinan-gold nanocluster fluorescent probe is 140~160 nm, and the lentinan-gold nanocluster fluorescent probe retains the triple helix structure of lentinan.

[0032] Thirdly, the present invention provides the application of the lentinan-gold nanocluster fluorescent probe in the preparation of tumor imaging products, tumor treatment products, or integrated tumor diagnosis and treatment products.

[0033] The lentinan-gold nanocluster fluorescent probe of the present invention possesses fluorescence imaging capability, peroxidase-like activity and anti-tumor bioactivity. It can achieve tumor enrichment through enhanced penetration and retention effects on tumor sites, and can be used for tumor imaging, tumor treatment or integrated tumor diagnosis and treatment.

[0034] This invention has at least one of the following beneficial effects: This invention utilizes amino-modified lentinan (LNT-NH2) and glutathione-modified gold nanoclusters (Au-GSH) as raw materials, and covalently couples them under mild conditions via a HATU / HOBT / DIEA system to prepare a lentinan-gold nanocluster fluorescent probe (LNT-Au). This preparation process avoids strong acids, strong bases, and high temperatures, effectively preserving the natural triple helix conformation of lentinan and thus maintaining its inherent antitumor biological activity. Furthermore, the prepared LNT-Au fluorescent probe has a uniform particle size, with an average size of approximately 150 nm, which is beneficial for tumor enrichment through the enhanced penetration and retention effect (EPR effect). In the tumor microenvironment, the inherent antitumor activity of lentinan can synergistically interact with the peroxidase-like activity-induced tumor cytotoxicity of Au-GSH. Simultaneously, the probe possesses a stable fluorescence signal, enabling in vivo tracking and imaging. The method of this invention has a simple preparation process and good reproducibility. The product obtained has both therapeutic and tracing functions and has broad application prospects in the fields of integrated tumor diagnosis and treatment, bioimaging and nanomedicine. Attached Figure Description

[0035] Figure 1 The zeta potentials of LNT-NH2, Au-GSH, and LNT-Au in aqueous solution in Example 2 are shown.

[0036] Figure 2 The images show the UV-Vis absorption spectra of LNT-NH2, Au-GSH, and LNT-Au in aqueous solution in Example 3.

[0037] Figure 3 The experiment with LNT-Au and Congo Red in Example 4.

[0038] Figure 4 This is a characterization of the catalytic activity of LNT-Au peroxidases based on UV-Vis absorption spectroscopy in Example 5.

[0039] Figure 5 This is a transmission electron microscope image of LNT-Au in Example 6.

[0040] Figure 6 This is an in vivo imaging image of tumor-bearing mice 24 hours after LNT-Au was injected via the tail vein in Example 7. Detailed Implementation

[0041] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0042] The amino-modified lentinan (LNT-NH2) and glutathione-modified gold nanoclusters (Au-GSH) in the following examples were synthesized in the laboratory previously.

[0043] The synthesis method of aminated lentinan (LNT-NH2) is as follows: Lentinan (LNT) is dissolved in DMSO at a concentration of 15 mg / mL, and the pH is adjusted to 8.5. Succinic anhydride (30% of the dry weight of LNT) is dissolved in 1 mL of DMSO and slowly added to the sugar solution. The pH is maintained at 8.5 during the reaction, and after the reaction, the pH of the sugar solution is adjusted to 6.5. After dialysis and concentration, the sugar solution is precipitated with 70% ethanol, washed three times with anhydrous ethanol by centrifugation, dialyzed using an 8000 Da dialysis bag, and then freeze-dried to obtain LNT-COOH. 500 mg of LNT-COOH was dissolved in DMSO to a concentration of 10 mg / mL. HATU (38.31 mg, 0.1 mmol), HOBT (14.3 mg, 0.1 mmol), and DIEA (26.04 mg, 0.2 mol) were added. After stirring for 3 min, tert-butyloxycarbonyl hydrazine (H2N-NHBOC) (100 mg, 0.62 mmol) was added, and the mixture was stirred for 2 h. Then, 0.5 mL of 50% trifluoroacetic acid was added, and the mixture was stirred for 1 h. After the reaction was complete, the solution was dialyzed through an 8000 Da dialysis bag, concentrated, and freeze-dried to obtain LNT-NH2.

[0044] The synthesis method of glutathione-modified gold nanoclusters (Au-GSH) is as follows: 10 mg of HAuCl4... H₂O (1.3 mM) was dissolved in 20 mL of ultrapure water and stirred. Then, 32 mg of glutathione (5.2 mM) was added, producing a microemulsion suspension. After vigorous stirring for 5 min, 20 mL of freshly prepared NaBH₄ solution (10 mg, 13 mM) was rapidly added. The color immediately turned dark brown, indicating the formation of gold clusters. Stirring was continued for 24 h. Purification and concentration were achieved using a 50 mL Amicon 3K centrifuge filter.

[0045] Example 1 This embodiment provides a method for preparing a lentinan-gold nanocluster fluorescent probe, comprising the following steps: Step 1: Dissolution of glutathione-modified gold nanoclusters (Au-GSH): Dissolve 100 mg of concentrated Au-GSH in 50 mL of DMSO and stir at room temperature for 30 min until completely dissolved, ensuring that Au-GSH is fully dispersed in the solvent to obtain a solution.

[0046] Step 2: Add HATU, HOBT, and DIEA: Add 38.31 mg (0.1 mmol) of condensing agent HATU, 14.3 mg (0.1 mmol) of auxiliary agent HOBT and 26.04 mg (0.2 mmol) of alkaline catalyst DIEA to the solution in sequence, stir for 3 min to ensure that all reagents are completely dissolved and reacted fully to obtain the reaction solution.

[0047] Step 3: Add amino-modified lentinan (LNT-NH2): Add 100 mg of LNT-NH2 to the reaction solution and stir at room temperature for 2 h to ensure that the aminoated lentinan is fully combined with Au-GSH.

[0048] Step 4: Dialysis and Concentration After the reaction was complete, the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 50,000 Da to remove unreacted reagents and small molecule impurities. The dialysis time was 72 h. After dialysis, the solution was concentrated at a concentration ratio of 1:5.

[0049] Step 5: Freeze-drying The concentrated solution was freeze-dried at -80℃ for 48 h to obtain the lentinan-gold nanocluster fluorescent probe (LNT-Au).

[0050] Example 2 The successful preparation of LNT-Au in Example 1 was verified by the change in zeta potential. The specific steps are as follows: Step 1: Prepare 5 mg / mL LNT-NH2 solution, Au-GSH solution and LNT-Au solution respectively.

[0051] Step 2: The zeta potentials of LNT-NH2 solution, Au-GSH solution and LNT-Au solution were determined using a particle size potential analyzer (Zetasizer Pro, Malvern Panaco LTD.).

[0052] Experimental results are as follows Figure 1 As shown, Au-GSH exhibits a lower Zeta potential. After amide condensation, the Zeta potential of LNT-Au differs significantly from that of LNT-NH2, indicating that LNT-Au was successfully prepared.

[0053] Example 3 The successful preparation of LNT-Au in Example 1 was verified by UV-Vis absorption spectroscopy. The specific steps are as follows: Step 1: Prepare 5 mg / mL LNT-NH2 solution, Au-GSH solution and LNT-Au solution respectively.

[0054] Step 2: Measure the UV-Vis absorption spectrum of the solution using a UV-Vis spectrophotometer. Experimental results are as follows Figure 2 As shown, LNT-Au exhibits similar characteristic absorption to Au-GSH, indicating the successful preparation of the LNT-Au fluorescent probe.

[0055] Example 4 The retention of the LNT-Au triple helix structure in Example 1 was verified using the Congo red assay. The specific steps are as follows: Step 1: Prepare a 1 mg / mL LNT-Au sample solution.

[0056] Step 2: Prepare an 89 μmol / L Congo red solution.

[0057] Step 3: Prepare a 4 mol / L sodium hydroxide solution.

[0058] Step 4: Take 0.5 mL of LNT-Au sample solution and 0.5 mL of Congo red solution and mix them. Add a certain amount of sodium hydroxide solution to make the sodium hydroxide concentration of the system 0, 0.05, 0.1, 0.2, 0.3 and 0.5 mol / L respectively, and shake gently to mix.

[0059] Step 5: After reacting for 15 minutes in the dark, the maximum absorption wavelength of the reaction solution is measured using a UV-Vis spectrophotometer.

[0060] Experimental results are as follows Figure 3 As shown, LNT-Au can form a complex with Congo red, resulting in a red shift in absorption wavelength compared to Congo red. Furthermore, within a certain sodium hydroxide concentration range, the maximum absorption wavelength decreases sharply, indicating that LNT-Au retains its triple helix structure.

[0061] Example 5 The peroxidase-like activity of LNT-Au in Example 1 was verified by the TMB colorimetric reaction. The specific steps are as follows: Using 80 mM TMB and 1 mM H2O2 as substrates, 2 mg / mL LNT-Au was added, and the reaction was carried out in the dark for 5 min before the UV-Vis absorption spectrum was measured.

[0062] Experimental results are as follows Figure 4 As shown, the reaction solution turned distinctly blue and exhibited a clear absorption peak at 652 nm, indicating the presence of a large number of hydroxyl radicals in the solution reacting with TMB, which originates from the catalytic effect of LNT-Au on H2O2. This demonstrates that LNT-Au possesses good peroxidase-like activity.

[0063] Example 6 The morphology of LNT-Au in Example 1 was observed using transmission electron microscopy. The specific steps are as follows: LNT-Au at a concentration of 2 mg / mL was added to a copper grid and observed using a transmission electron microscope.

[0064] Experimental results are as follows Figure 5 As shown, a large number of black nanoparticles are uniformly distributed with an average size of about 150 nm, which is much larger than the average size of Au-GSH (about 2 nm). This confirms the successful construction of the LNT-Au fluorescent probe. On the other hand, this size is conducive to achieving tumor enrichment through the enhanced penetration and retention effect (EPR effect) of tumors.

[0065] Example 7 The enrichment ability of LNT-Au in tumor tissue in Example 1 was verified using near-infrared II in vivo imaging. The specific steps are as follows: LNT-Au was injected into tumor-bearing mice via the tail vein at a dose of 30 mg / kg, and the backs of the mice were imaged using a near-infrared 2D in vivo imaging system.

[0066] Experimental results are as follows Figure 6 As shown, at 24 h, there was a relatively obvious fluorescent signal at the tumor site in mice, indicating that LNT-Au can be significantly enriched at the tumor tissue, providing a strong premise for its therapeutic effect at the tumor site.

[0067] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a lentinan-gold nanocluster fluorescent probe, characterized in that, Includes the following steps: A lentinan-gold nanocluster fluorescent probe was prepared by covalently coupling the amino-modified lentinan and glutathione-modified gold nanoclusters under mild conditions.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the amino-modified lentinan to the glutathione-modified gold nanoclusters is 0.8~1.2:0.8~1.

2.

3. The preparation method according to claim 1, characterized in that, The preparation method of the amino-modified lentinan includes the following steps: Lentinan was dissolved in an organic solvent to obtain a lentinan solution, and the pH of the lentinan solution was adjusted to 8.5-9. Succinic anhydride was dissolved in an organic solvent to obtain a succinic anhydride solution; the succinic anhydride solution was added to the lentinan solution for reaction, and the pH of the system was maintained at 8.5-9 during the reaction. After the reaction was completed, the pH of the system was adjusted to 6.5-7. The product was dialyzed, concentrated, precipitated, washed and dried to obtain LNT-COOH. The LNT-COOH was dissolved in an organic solvent, and then HATU, HOBT and DIEA were added to react. H2N-NHBOC was then added to carry out an amination reaction. After the reaction was completed, trifluoroacetic acid was added and stirred. The product was dialyzed, concentrated and dried to obtain amination-modified lentinan.

4. The preparation method according to claim 1, characterized in that, The method for preparing the glutathione-modified gold nanoclusters includes the following steps: HAuCl4 H2O was dissolved in water, and glutathione was added to produce a microemulsion suspension. After vigorous stirring, NaBH4 solution was quickly added and stirring continued. The product was then filtered, purified, and concentrated to obtain glutathione-modified gold nanoclusters.

5. The preparation method according to claim 1, characterized in that, The preparation method includes the following specific steps: Glutathione-modified gold nanoclusters were dissolved in dimethyl sulfoxide to obtain an Au-GSH solution. A condensing agent, an auxiliary agent, and an alkaline catalyst were added sequentially to the Au-GSH solution. After stirring and activation, amino-modified lentinan was added to carry out the reaction. After the reaction was completed, the solution was dialyzed, concentrated, and freeze-dried to prepare a lentinan-gold nanocluster fluorescent probe.

6. The preparation method according to claim 5, characterized in that, The concentration of the Au-GSH solution is 1.5~2.5 mg / mL; the molar ratio of the condensing agent, auxiliary agent and alkaline catalyst is 0.1:0.1:0.

2.

7. The preparation method according to claim 5, characterized in that, The condensing agent is HATU, the auxiliary agent is HOBT, and the alkaline catalyst is DIEA; The dialysis was performed using a dialysis bag with a molecular weight cutoff of 50,000 Da; The reaction time after adding the aminoated lentinan is 1.5 to 2.5 h.

8. A lentinan-gold nanocluster fluorescent probe, characterized in that, It is obtained by the preparation method described in any one of claims 1 to 7.

9. The lentinan-gold nanocluster fluorescent probe according to claim 8, characterized in that, The average particle size of the lentinan-gold nanocluster fluorescent probe is 140~160 nm, and the lentinan-gold nanocluster fluorescent probe retains the triple helix structure of lentinan.

10. The use of the lentinan-gold nanocluster fluorescent probe according to any one of claims 8 to 9 in the preparation of tumor imaging products, tumor treatment products, or integrated tumor diagnosis and treatment products.

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